Recently, multiplexed methods have been developed suitable for genome analysis, detection of polymorphisms, molecular karyotyping, and gene-expression analysis, where the genetic material in a sample is hybridized with a set of anchored oligonucleotide probes. Understanding the results, displayed when labeled subsequences in the target bind to particular probes, is based on decoding the probe array to determine the sequence of the bound members.
Embodiments of such arrays include spatially encoded oligonucleotide probe arrays, where the array is placed on a chip and particular probes are placed in designated regions of the chip. Also included are particle or microbead arrays, designated random encoded arrays, where the oligonucleotide probes are bound to encoded beads, and where the encoding identifies the probe bound thereto. See, e.g., U.S. application Ser. No. 09/690,040 Such arrays allow one to conduct high-throughput multiplexed hybridization involving thousands of probes on a surface.
One problem in multiplexed analysis is in interpreting the results. In a conventional multiplexed “reverse dot blot” hybridization assay, the target oligonucleotide strands are labeled, and the labeling is detected, post-hybridization to the probe array, to ascertain binding events. The reliability of the final computational interpretation of the data depends on understanding the errors due to unintended interactions among targets and probes, as probes and targets are multiplexed.
The standard approach to the thermodynamic analysis of nucleic acid hybridization invokes interactions between adjacent (“nearest-neighbor”) base-pairs to account for the thermodynamic stability of the duplex under given experimental conditions including temperature, ionic strength, and pH (see, e.g., Cantor & Smith, “Genomics”, Wiley Interscience, 1999). A variety of commercial software packages for probe and primer design invoke this model to estimate the thermodynamic stability, or equivalently, the “melting temperature”, Tm, usually defined as the temperature at which half of a given number of nucleic acid duplexes have separated into single strands—a phenomenon also referred to as “denaturing” or “de-annealing.” The melting temperature of a probe or primer—more precisely, the duplex formed by the probe or primer and a complementary single oligonucleotide strand—represents the single most widely used parameter to guide the design of probes and primers in assays involving hybridization. Many commercial software packages are available for this purpose, e.g., OLIGO, VISUALOMP, PRIMERSELECT, ARRAY DESIGNER, PRIMER3, and others.
Duplex stability is significantly affected by the presence of mismatched bases, so that even a single point mutation can substantially lower the melting temperature (FIG. 1). This phenomenon provides the basis for the standard method of detecting such a mutant or polymorph in a designated location within a target sequence using hybridization analysis: a pair of probes, one member complementary to the normal target composition at the designated location, the other complementary to an anticipated variant, are permitted to interact with the target. For example, in a “reverse dot blot” or READ™ assay format, both probes are immobilized and permitted to capture a portion of a labeled target molecule of interest that is placed in contact with the probes. It is the objective of such an analysis to optimize discrimination between the signal associated with the “perfectly matched (PM)” probe having the sequence that is perfectly matched to the target sequence and the signal associated with the “mismatched (MM)” probe having the sequence that deviates from that of the target sequence in the designated location. It is desirable for the signal intensity to indicate the amount of captured target; an example would be target-associated fluorescence, where a higher intensity signal indicates more capture. For a single pair of PM/MM probes, optimal discrimination would be ensured by selecting the temperature to fall between the respective melting temperatures, TmPM and TmMM<TmPM of perfectly matched and mismatched probes (FIG. 1). However, in multiplexed reactions, involving two or more PM/MM probe pairs, no such choice of a single temperature generally will be possible. In addition, multiplexed mutation analysis generally has to account for the possibility of cross-hybridization between other than cognate probes and targets, a possibility that exists whenever probes or targets display a sufficiently high degree of homology so as to interact with sufficient strength so as to stabilize a duplex under given experimental conditions.
Another source of error arises from competitive hybridization, where a target consists of possibly two distinct subsequences mA and mB, which can be characterized by separately hybridizing the target with either a mixture of specific probes mmA and control probes mmA, or a mixture of specific probes pmB and control probes mmB, respectively. In either case the ratio of specific signal to the control signal, obtained from each separate experiment, indicates how often either message is present. On the other hand, contrary to one's expectations, if the two messages were queried by ratios of the respective signals in a multiplexed experiment consisting of all four probes pmA, mmA, pmB, and mmB, one finds these ratios to differ from their values in the earlier experiments and by amounts that cannot simply be explained by the statistical noise. In particular, if one of the ratio values decreases severely, the resulting false negative errors can become significant enough to affect the multiplexed assay results. The negative effects can worsen as the number of multiplexed probes for a target increases.
There is a need for a method to correct for such errors, and also for assay design methods and probe selection, which will avoid or minimize such errors.